1.1 Field of the Invention
The present invention relates generally to the cyclooxygenases and their roles in cancer and inflammation. More particularly, this invention pertains to methods and articles of manufacture for detecting and measuring COX-2 activity by detecting and measuring COX-2 specific enzymatic products including glyceryl-prostaglandins.
1.2 Description of the Related Art
COX, or prostaglandin endoperoxide synthase enzyme (cyclooxygenase, COX, EC 1.14.99.1), catalyzes the conversion of arachidonic acid to prostaglandin (PG) H2. Two isoforms of COX are known, COX-1 and COX-2. COX-1 is constitutively expressed. COX-2, however, is inducible in a variety of cells, especially those of the central nervous and immune systems (Masferrer et al. 1994, Proc. Natl. Acad. Sci. USA 91:3228-3232; Vane et al. 1994, Proc. Natl. Acad. Sci. USA 91:2046-2050; Kennedy et al. 1993, Biochem. Biophys. Res. Commun. 197:494-500). Certain changes in COX-2 activity are associated with a variety of human inflammatory diseases. These diseases include, but are not limited to, acute appendicitis, asthma, myocardial infarction, certain immunological disease processes, infection, malignancy, endotoxemia and reperfusion injury. In addition, inappropriate COX-2 expression or over-expression is associated with certain types of cancers, including, but not limited to, carcinoma of the colon, rectum, stomach, esophagus, lung, and skin. The amount of COX-2 expression is related to the cancer stage and grade (Fosslien, E, et al. 2000, Ann. Clin. Lab. Sci. 30:3-21). COX-2 has become a major pharmaceutical target for developing treatments for these and other diseases. Methods of detecting and measuring COX-2 activity are highly desired.
Yu et al. (1997) J. Biol. Chem. 272:21181-21186, describes the enzymatic conversion of arachidonyl ethanolamide (anandamide, AEA), to PGE2-ethanolamide in cell lines expressing COX-2 but not COX-1.
U.S. Pat. No. 5,543,297 to Cromlish et al., describes measuring total COX activity (COX-1 activity and COX-2 activity) in separate samples, with and without a COX-2 specific inhibitor, and then indirectly estimating COX-2 specific activity by subtracting the total COX activity observed with the inhibitor from the total COX activity observed without the inhibitor. One major weakness of this method is that the dynamics of enzymatic inhibition change based upon numerous variables including time, temperature, concentration, specificity, sample preparation, etc.
U.S. Pat. No. 5,475,021 to Marnett et al. describes a method of measuring the activity of purified COX-2 by measuring O2-uptake during catalysis. This method requires purification of the enzyme.
U.S. Pat. No. 6,045,773 to Isakson et al., describes a method for measuring COX-2 expression in a mammal by administering a positron-emitting radioisotope-labeled COX-2 selective binding agent to the mammal and then detecting the label by positron-emission tomography (PET). Weaknesses of this method include the invasive nature and expense of PET equipment. In addition, the method only localizes COX-2 protein but does not detect or measure activity.
What is needed, then, is a less-invasive, method of selectively detecting and measuring COX-2 activity in biological samples without the need to purify the enzyme.